Cryptococcus neoformans is a human pathogenic fungus that causes life-threatening meningoencephalitis in immunocompromised individuals. One important aspect of cryptococcosis is the ability of C. neoformans cells to cross the blood-brain barrier. While the processes involved in central nervous system (CNS) penetration have been elucidated over the past few years, the methods by which C. neoformans induces these processes are unknown. Interestingly, C. neoformans has two mating types (a and a);however the vast majority of clinical isolates are a mating type. While no differences in virulence are observed in individual infections with congenic strains in the most commonly pathogenic variety of Cryptococcus during coinfection of a and a strains simultaneously, the a cells predominate in the CNS. By comparing the a and a cells in individual and coinfections we have the opportunity to identify virulence factors specifically required for entry into the CNS. Because C. neoformans cells sense and respond to cells of the opposite mating partner using pheromones, in preliminary experiments we disrupted pheromone sensing in both a and a cells and discovered that a cells are inhibited from entering the brain by pheromone signaling. We hypothesize that pheromone signaling plays a central role in C. neoformans entry into the CNS and that the downstream targets of this pheromone signaling affect the interaction of C. neoformans with the blood-brain barrier. We propose to test these hypotheses by perturbing in vivo pheromone signaling using mutant strains altered in either pheromone sensing or pheromone production. The virulence of these mutant strains will be examined in individual and coinfections and used to help identify the downstream targets of this pheromone signaling by microarray analysis. This information will then be used to examine changes in cellular traits that affect CNS entry. Furthermore, pheromone levels will be examined during the infectious process to define when pheromone signaling is likely to be important for virulence potential. These studies will allow the identification of key components of CNS penetration by Cryptococcus and provide a foundation for treatment strategies designed to reduce CNS penetration to alleviate disease symptoms and allow for increased exposure to antifungal drugs.